Diffuse reflectance spectra, which are indicative of the absorption and scattering properties of cells and/or tissues, are sensitive to a number of important biological molecules. In cells and/or tissues, absorption is due at least in part to the presence of various biological molecules, such as proteins, carotenoids, and hemoglobin, and scattering is attributed inter alia to the size and density of intracellular and extracellular structures. Diffuse reflectance spectroscopy has therefore been investigated as a possible approach to diagnosing early pre-cancerous and cancerous changes in such cells and/or tissues (Thueler et al. (2003) 8 J Biomed Opt 495-503; Muller et al. (2001) 40 Appl Opt 4633-46; Palmer et al. (2003) 50 IEEE Trans Biomed Eng 1233-42; Finlay & Foster (2004) 31 Med Phys 1949-59; Georgakoudi et al. (2002) 62 Cancer Res 682-687, 2002). However, due to the complex interplay between absorbers and scatterers in cells and/or tissues, it can be difficult to relate a measured diffuse reflectance spectrum to the underlying physical features of the cells and/or tissues.
The illumination/collection geometry of the probe that is employed can be an important aspect of cell/tissue optical spectroscopic measurements in that it can affect sensitivity to the optical properties (absorption and scattering coefficients), sensing volume, and signal to noise (Mourant et al. (1997) 36 Appl Opt 5655-5661; Zhu et al. (2003) 8 J Biomed Opt 237-247; Pogue & Burke (1998) 37 Appl Opt 7429-36). There are numerous possible probe designs to select from for a given biomedical application.
Specialized probe designs have been previously shown to be useful in characterizing tissue properties from fluorescence (Pogue & Burke (1998) 37 Appl Opt 7429-36; Pfefer et al. (2004) 42 Med Biol Eng Comput 669-73; Pfefer et al. (2005) 10 J Biomed Opt 44016; Zhu et al. (2005) 10 J Biomed Opt 024032; Quan & Ramanujam (2004) 29 Opt Lett 2034-2036) and diffuse reflectance measurements (Mourant et al. (1997) 36 Appl Opt 5655-5661; Amelink et al. (2004) 29 Opt Lett 1087-1089). For example, Mourant et al. (36 Appl Opt 5655-5661, 1997; hereinafter, “Mourant”) discloses that at a source-detector separation of approximately 1.7 mm, the diffuse reflectance collected was insensitive to the scattering coefficient.
Thus, the measured diffuse reflectance could be directly related to the absorption coefficient. Mourant further discloses that for a source-detector separation of 1.7 mm, this relationship is valid for absorption coefficients in the range of 0-0.86 cm−1 and reduced scattering coefficients in the range of 7-21 cm−1. Using this relationship, the authors were able to extract the concentration of Direct Blue dye from a phantom with errors of 20% or less. This method furthermore required no a priori information about the absorbers and scatterers present in the medium.
However, the error for the reported probe is potentially too great to allow the disclosed probe to be employed for sensitive medical applications, and it is not valid for optical properties typical of tissue in the UV-visible wavelength range. Additionally, Mourant does not optimize the geometry of the fiber optic probe, instead simply testing only the operation of probes with a different separation between source and detector fibers. What are needed, then, are methods for testing various parameters of fiber optic probes for spectroscopic measurements that can be used to optimize probe geometries for applications for which enhanced accuracy is important.
To address this need, the presently disclosed subject matter provides methods for optimizing a fiber optic probe geometry for spectroscopic measurement. Such methods are useful for identifying probe geometries that can be employed for measuring optical properties of cells, tissues, or other turbid media.